The bovine spongiform encephalopathy (BSE) agent has been transmitted tohumans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can beexperimentally infected by BSE and have been potentially exposed to naturalBSE; however, whether BSE can be transmitted to small ruminants is notknown. Based on the particular biochemical properties of the abnormal prionprotein (PrPsc) associated with BSE, and particularly the increaseddegradation induced by proteinase K in the N terminal part of PrPsc, we havedeveloped a rapid ELISA designed to distinguish BSE from other scrapiestrains. This assay clearly discriminates experimental ovine BSE from otherscrapie strains and was used to screen 260 transmissible spongiformencephalopathy (TSE)–infected small ruminant samples identified by theFrench active surveillance network (2002/2003). In this context, this testhas helped to identify the first case of natural BSE in a goat and can beused to classify TSE isolates based on the proteinase K sensitivity ofPrPsc.

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Analysis of Nor98 Isolates

The typing test was used to analyze 18 sheep isolates from Norway (Table 3).Ratios were almost impossible to calculate because of the large decrease insignal in A´ conditions, as shown in Figure 5, panel A for 3 isolates. Only1 sample (Lavik) showed characteristics of a conventional scrapie isolate,providing an A/A´ ratio of 0.84 (Figure 5, panel A), a normalized ratio of0.11, and a Western blot profile close to that of a French scrapie isolate(Figure 4, panel B, lanes 3 and 9; Figure 5, panel B, lanes 2 and 4). Othersamples had a pattern that included a 12-kDa band (Figure 5, panel B)(19,22,34), characteristic of the Nor-98 strain.

After adapting the conditions of the PK treatment in the second set ofmeasurements (A´ conditions), we observed (see legend, Figure 6) a muchlower A/A´ ratio for those Nor-98, which enables discrimination of highlysensitive PK samples (nos. 24 and 26, Appendix Figure 2 and Table 3) tomildly sensitive PK samples (nos. 8, 11, 16, and 22).

Discussion

When this study was initiated, no case of natural BSE in small ruminants wasrecorded, and only a few experimental ovine BSE samples were available, allbelonging to the same PrP genotype (ARQ/ARQ), and mainly from a firstpassage. The possible impact of the genotype, the route of infection, andthe number of passages on the biochemical properties of PrPres associatedwith the BSE strain are poorly understood. Now, further data suggest that,at least during the second passage in sheep, the biochemical properties(glycoform pattern in brain) of the BSE agent are unchanged (35,36). In thisstudy using our ELISA, small ruminant BSE samples clearly behaveddifferently from conventional scrapie samples. However, slight differencesmay exist (see ARQ/ARQ vs. ARR/ARR genotype in Appendix Figure 1). We do notknow whether these findings reflect differences in the PK sensitivity ofPrPres associated with these genotypes or the influence of differenttissues.

The main difficulty encountered for the development of a typing test isevaluation of its specificity and sensitivity. In the current study, weunambiguously identified all 37 experimental ovine BSE samples from 25sheep, including 10 from a second passage. There are few data describing themolecular features of PrPres associated with experimental BSE in goats(37,38). In the framework of the French scrapie strain-typing network, 18goats were analyzed by this ELISA, and 2 appeared compatible withexperimental ovine and caprine BSE. One of them (Ch636), when analyzed withother molecular typing tests, appeared indistinguishable from experimentalBSE and was later confirmed as the first natural case of BSE in a goat (1),after experimental transmission in wild-type and transgenic mice. The secondBSE compatible sample (TR041528) was later clearly identified as a case ofatypical scrapie as defined by its migration pattern (34). All these datasuggest a good sensitivity for our test, which unambiguously identified allcases of experimental BSE in the sheep and goats tested, as well as the onlynatural case identified to date in a goat.

Another key point during the development of this test was to ensure goodreproducibility because this parameter clearly influences both sensitivityand specificity. Ratios obtained for the classic scrapie control were highlyreproducible, whereas ratios measured for the experimental BSE in sheep andthe intermediate scrapie control varied much more, leading to an overlap ofthe 95% confidence interval (Table 1). To minimize interassay variations,the ratio obtained for each unknown sample was thus normalized by taking asreference the ratio measured for the ovine BSE sample (Figure 2, panel C,and Figure 3) in the same experiment. This enabled us to define the range ofnormalized ratio compatible with BSE as the mean of experimental ovine BSE ±2s on the basis of reproducibility experiments recorded in Table 1. Thisrange was experimentally determined between 0.7 and 1.3, leading to 3categories for field samples: conventional scrapie (ratio <0.7), compatiblewith BSE (0.7 1.3).

Only 10 (3.8%) of the 260 samples analyzed in the framework of the Frenchepidemiologic surveillance network during 2002–2003 gave a ratio compatiblewith BSE. Of the 10 BSE suspected samples, only 1 goat sample (Ch636) waslater confirmed as a true natural BSE case (1). This result indicates thatthe specificity of this test is not that good because 9 false-positiveresults were recorded in 260 samples (specificity 96.5%). However, the testappears useful since it excluded the presence of BSE for most field samples,thus restricting the use of more specific but time-consuming methods, likeexperimental transmission in mice, to a small number of isolates. Moreover,in a single screening, this test classified all TSE-infected isolates as afunction of their PK resistance and thus provided a rapid classification ofsheep isolates according to this criterion. The test could also be modified,by adjusting the range of PK sensitivity, to classify Nor-98 isolates.

All these data demonstrate that this ELISA-based typing test is suitable fora routine analysis of field samples, as assessed by the positive evaluationfrom the European Commission as one of the tests recommended to identify thepossible presence of BSE in small ruminant flocks(http://eur-lex.europa.eu/LexUriServ/site/en/oj/2005/l_010/l_01020050113en00090017.pdf). These typing tests are mainly designed to identify the BSEstrain in small ruminant flocks. They are performed exclusively in nationalreference laboratories and based on Western blot techniques. In thiscontext, the present ELISA is one of the secondary tests to be used toconfirm BSE suspicion. We believe it will help clarify the status of theseunusual isolates.

Abstract Prion diseases are characterized by a long incubation period. Inscrapie, sheep may incubate and spread the infection for several yearsbefore clinical signs evolve. We have previously studied the occurrence ofsubclincal infection in the brain. Now, we have studied the occurrence ofsubclinical infection in the brain and several lymphoid tissues in twoscrapie-affected Icelandic sheep flocks by immunohistochemistry for PrPSc, amolecular marker for infectivity, and correlated this with results of PrPgenotyping. At culling, one flock had one confirmed scrapie case, while theother flock had two. Analysis of 106 asymptomatic sheep by immunostainingfor PrPSc revealed that the incidence of subclinical infection was 58.3% inone flock and 42.5% in the other. PrPSc was only detected in lymphoidtissues. The youngest positive sheep were 4 months old. PrP genotypingshowed that over 90% of the sheep were of a genotype which is moderatelysensitive to infection and may delay neuroinvasion. Our results show thatasymptomatic sheep may spread the infection during the long incubationperiod of several years, which constitutes an important obstacle in theeradication of scrapie. Our findings indicate that contamination of theenvironment plays an important part in sustaining the infection.

There were 27 scrapie infected and source flocks with open statuses (Figure3) as of January 31, 2008. Two new source flocks and one new infected flockwere reported in January (Figure 4) with a total of 22 reported for FY 2008(Figure 5). ....

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Positive Scrapie Cases

As of January 31, 2008, 58 new scrapie cases have been confirmed andreported by the National Veterinary Services Laboratories (NVSL) in FY 2008(Figure 7). Of these, 52 were field cases and 6* were Regulatory ScrapieSlaughter Surveillance (RSSS) cases (collected in FY 2008 and reported byFebruary 20, 2008). There were 8 positive cases for January which aredepicted in Figure 8. Seventeen cases of scrapie in goats have beenconfirmed by NVSL since implementation of the regulatory changes in FY 2002(Figure 9). The most recent positive goat cases were from the SAME HERD andWERE BOTH CONFIRMED IN JANAURY 2008.

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Caprine Scrapie Prevalence Study (CSPS)

CSPS was initiated in May 2007 to estimate the national prevalance ofscrapie in adult goats at slaughter. If no scrapie is found we will be ableto conclude that the prevalence in goats is greater than zero and less than0.1 percent. AS of January 31, 2008, 2,942 goats have been sampled forscrapie testing (1,515 in FY 2007 and 1,427 in FY 2008). Collection numbersby quarter in FY 2008 is shown in Chart 8. To date, no goats have testedpositive for scrapie as part of this surveillance program. HOWEVER, THREEPOSITIVE GOATS have been identified this fiscal year through fieldinvestigations. One was a clinical suspect submitted for testing and THEOTHER TWO WERE MEMBERS OF THE OF THE BIRTH HERD OF THE CLINICAL CASE.

INFECTED AND SOURCE FLOCKS AS of August 31, 2007, there were 33 scrapieinfected and source flocks with open statuses (Figure 3). Five new sourceflocks and one new infected flock were reported n August (Figure 4) with atotal of 64 reported for FY 2007(Figure 5).

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IN FY 2007 TWO FIELD CASES, ONE VALIDATION CASE, AND TWO RSSS CASES WERECONSISTENT WITH NOR-98 SCRAPIE. ...

> If atypical scrapie is found in the United States, an additional controlprogram may be necessary

> but it is likely that no changes in the current control program will beneeded.

http://www.usaha.org/committees/reports/2006/report-scr-2006.pdf

>>>For the period of time from January 1, 2005, until October 15, 2005,there were 23 instances of discrepancies in results from 35 flocks. Of those23 instances, 14 were caused by laboratory error (paperwork or samplemix-up), 3 results from field error, 5 were not completely resolved, and 1originated from the use of a non-approved laboratory for the first test. Asa result of inconsistencies, one laboratory’s certification was revoked byAPHIS-VS. <<<

http://www.usaha.org/committees/reports/2005/report-scr-2005.pdf

SCRAPIE TYPICAL AND ATYPICAL USA

http://nor-98.blogspot.com/

http://scrapie-usa.blogspot.com/

> -------- Original Message --------> Subject: USA BIO-RADs INCONCLUSIVEs> Date: Fri, 17 Dec 2004 15:37:28 -0600> From: "Terry S. Singeltary Sr."> To: susan_berg@bio-rad.com>>>> Hello Susan and Bio-Rad,>> Happy Holidays!>> I wish to ask a question about Bio-Rad and USDA BSE/TSE testing> and there inconclusive. IS the Bio-Rad test for BSE/TSE that complicated,> or is there most likely some human error we are seeing here?>> HOW can Japan have 2 positive cows with> No clinical signs WB+, IHC-, HP- ,> BUT in the USA, these cows are considered 'negative'?>> IS there more politics working here than science in the USA?>> What am I missing?>>>> -------- Original Message --------> Subject: Re: USDA: More mad cow testing will demonstrate beef's safety> Date: Fri, 17 Dec 2004 09:26:19 -0600> From: "Terry S. Singeltary Sr."> snip...end>>> Experts doubt USDA's mad cow results

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WELL, someone did call me from Bio-Rad about this,however it was not Susan Berg.but i had to just about take a blood oath not to revealthere name. IN fact they did not want me to even mentionthis, but i feel it is much much to important. I have omittedany I.D. of this person, but thought I must document this ;

at approx. 14:00 hours 12/28/04, I had a very pleasantphone conversation with XXXX XXXXX about the USDAand the inconclusive BSE testing problems they seemto keep having. X was very very cautious as to speakdirectly about USDA and it's policy of not using WB.X was very concerned as a Bio-Rad official of retaliationof some sort. X would only speak of what other countriesdo, and that i should take that as an answer. I told XI understood that it was a very loaded question and Xagreed several times over and even said a political one.

my question;

Does Bio-Rad believe USDA's final determination of False positive,without WB, and considering the newatypical TSEs not showing positive with -IHC and -HP ???

ask if i was a reporter. i said no, i was with CJD Watchand that i had lost my mother to hvCJD. X did notwant any of this recorded or repeated.

again, very nervous, will not answer directly about USDA for fear ofretaliation, but again said X tellme what other countries are doing and finding, and thati should take it from there."very difficult to answer"

"very political"

"very loaded question"

outside USA and Canada, they use many different confirmatory tech. inhouse WB, SAF, along withIHC, HP, several times etc. you should see at severaltalks meetings (TSE) of late Paris Dec 2, that IHC- DOES NOT MEAN IT ISNEGATIVE. again, look whatthe rest of the world is doing.said something about Dr. Houston stating;any screening assay, always a chance for humanerror. but with so many errors (i am assumingX meant inconclusive), why are there no investigations, just falsepositives?said something about ''just look at the sheep that tested IHC- but werepositive''. ...

In June 2005, an inconclusive bovine spongiform encephalopathy (BSE) samplefrom November 2004, that had originally been classified as negative on theimmunohistochemistry test, was confirmed positive on SAF immunoblot (Westernblot). The U.S. Department of Agriculture (USDA) identified the herd oforigin for the index cow in Texas; that identification was confirmed by DNAanalysis. USDA, in close cooperation with the Texas Animal Health Commission(TAHC), established an incident command post (ICP) and began responseactivities according to USDA’s BSE Response Plan of September 2004. Responsepersonnel removed at-risk cattle and cattle of interest (COI) from the indexherd, euthanized them, and tested them for BSE; all were negative. USDA andthe State extensively traced all at-risk cattle and COI that left the indexherd. The majority of these animals entered rendering and/or slaughterchannels well before theinvestigation began. USDA’s response to the Texas finding was thorough andeffective.

On June 24, 2005, USDA informed FDA that a cow in Texas tested positive forBovine Spongiform Encephalopathy (BSE). Information provided by APHIS wasthat the BSE positive cow was born and raised in a herd in Texas and wasapproximately 12 years old. The animal was sampled for BSE at a pet foodplant in Texas on November 15, 2004, as part of USDA’s enhanced surveillanceprogram.

Secretary of Agriculture Ann M. Veneman resigns Nov 15 2004, three dayslaterinclusive Mad Cow is announced. June 7th 2005 Bill Hawks UnderSecretary forMarketing and Regulatory Programs resigns. Three days later same mad cowfound in November turns out to be positive. Both resignationare unexpected.just pondering...TSS

our computer department was working on a place holder we could postUSDA'sannouncement of any results. There are no results to be announcedtonight byNVSL, so we are back in a waiting mode and will post theUSDAannouncement when we hear something.